Comparison of one‐step with two‐step production of Bacillus atrophaeus spores for use as bioindicators

Abstract The production method of spores significantly influences the resistance of spores used as bioindicators (BI) in the validation of sterilization of packaging material surfaces in aseptic food manufacturing. Therefore, the standardization of the spore production method represents an important and desirable goal in industrial BI production to ensure reliable validation test results. Previously, we recommended a two‐step production approach for submerged spore production, in which the cultivation phase to obtain high cell mass was separate from the sporulation phase. In this work, a one‐step manufacturing process was investigated to reduce production complexity and facilitate standardization of spore production. It was found that one‐step BI production is technically possible but at the expense of spore yield. The two‐step manufacturing process can realize almost 10‐fold higher spore yields.


| INTRODUCTION
In the food and pharmaceutical industries, bioindicators (BI) consisting of spores of Bacillus atrophaeus or Geobacillus stearothermophilus are used to assess the inactivation performance of sterilization processes, for example, of packaging material treatment with liquid or vaporized hydrogen peroxide (FDA, 2007;McLeod et al., 2017; VDMA, 2021) before aseptic filling. It is known that the sporulation conditions can cause a significant change in the spore structure and, thus, in the resistance of the spores (Abhyankar et al., 2016;Bressuire-Isoard et al., 2016, 2018Isticato et al., 2020;Setlow, 2014). This means that the manufacturing method significantly influences the resistance of the BI, which may result in differences in the inactivation result and, thus, in uncertainties in the validity of sterilization process test results when using BI with variable and, most times, unknown resistances against the applied sterilization method. Therefore, standardization of BI production would be essential and a great step forward to ensure consistent quality and resistance of spores. Currently, state-of-the-art is that there are no agreed standardized BI production protocols, including the selection of nutrient contents, cell growth, and sporulation conditions.
We already showed that spore resistance can be influenced and predicted in submerged production or solid-state production on agar plates as a function of sporulation temperature and sporulation pH (Stier & Kulozik, 2020;Stier et al., 2021). Compared to the solid-state method, which typically results in a high degree of cell differentiation due to inconsistent conditions across and through the agar plate (Vlamakis et al., 2008), submerged production offers more consistent fermentation conditions. In our experiments on the submerged MicrobiologyOpen. 2022;5:è1332.
www.MicrobiologyOpen.com in its composition, was optimized to induce sporulation rapidly and simultaneously for all cells. This differs from other works that reported a one-step approach combining cultivation and sporulation. continued sporulation in the same medium and had to over inoculate the volume of the preculture for spore production into the same sporulation medium to achieve the desired high number of spores.
Since there was no washing step between cultivation and sporulation in the same medium required, this procedure can be described as a one-step method, although technically, there were steps transferring the cells at the end of cultivation into another bioreactor for sporulation.  did not over inoculate but combined the phase of cultivation and sporulation in a single production step simply by continuing the cultivation step to achieve sporulation in the same bioreactor using the same medium. This, however, resulted in a low spore yield.
Avoiding two separate production steps (i.e., the elimination of cultivation and sporulation in separate media) represents a substantial simplification of spore production. This way, it could have corresponding advantages in industrial BI production and facilitate standardization. The purpose of this work, therefore, was to test the one-step in a head-to-head comparison versus the two-step method.
To address the potential for optimization of spore production, we investigate the possibility of the one-step production process for its application in BI production. We combined this approach with testing the suitability of different established sporulation media types for different Bacillus species, modified, however, in their glucose contents to allow for more cell growth, on cultivating vegetative cells before the onset of sporulation and compared these results with cell growth in several cultivation media. The reason for increasing the glucose concentration in classical sporulation media with typically low glucose contents to induce starvation was that the vegetative cells should not sporulate immediately after cell growth when inoculated into the bioreactor to adapt to the sporulation conditions.
Finally, we reviewed the sporulation yield of B. atrophaeus in a one-step process in different sporulation media and compared the results with those obtained in our previous work (Stier & Kulozik, 2020), applying the two-step method with separated cultivation and sporulation steps.

| Strain
Spores of the strain B. atrophaeus ATCC 9372, which has been established as a hydrogen peroxide BI (FDA, 2007;McLeod et al., 2017;VDMA, 2021), was used as starting material. The spores were of the same production batch and stored at aliquots of 500 µl with a concentration of 10 7 colony-forming units (CFU)/ml at −80°C to ensure the initial characteristics of the spores did not change. The aliquots enabled the spores to be taken without thawing the rest of the spore suspension.

| Media
The following media were used for cultivation or, depending on the experiment, for the sporulation of B. atrophaeus. These media are typical growth media used in microbiology (Lysogeny Broth- LB comprised of casein peptone 10 g/L (for microbiology, Gerbu Biotechnik GmbH), yeast extract 5 g/L (for microbiology), and NaCl 5 g/L (ACS reagent, ≥99.0%).
TB contained casein peptone 12 g/L (for microbiology, Gerbu Biotechnik GmbH), yeast extract 24 g/L (for microbiology), K 2 HPO 4 9.4 g/L (ACS reagent, ≥98%), KH 2 PO 4 2.2 g/L (ACS reagent, ≥99%), Roth GmbH & Co. KG). This medium was not initially designed for the submerged growth of Bacillus spp. and was, therefore, not included in the submerged cultivation experiments in the beginning. However, the medium was applied in the sporulation experiments because, as described in more detail in the discussion, BSM was unsuitable for sporulation. Thus another sporulation medium had to be used for comparison. To adapt the medium for submerged sporulation, agaragar was not added in the media preparation.

| Cultivation of vegetative cells of B. atrophaeus
The aim was to compare the results from our previous work (Stier & Kulozik, 2020) applying two-step spore production (cultivation in TB and sporulation in DSM) with the one-step procedure according to   end of incubation to minimize the risk of regermination. The presence of living cells of the suspensions, which would falsify the spore concentration determination, was tested by phase-contrast microscopy. Vital cells were observed as motile unicellular or bicellular cells (Kearns & Losick, 2005) in the cultivation phase, whereas the remaining cells floated motionless in the suspension after the sporulation. Additionally, this observation is supported by the fact that the cells were cultured for 96 h in a sporulation medium that no longer offered adequate living conditions at a certain point in time, which is why the cells sporulated. Therefore, the presence of living cells is highly unlikely even without phase microscopic evidence.
For phase-contrast microscopic assessment of sporulation, the suspensions were concentrated by a factor of 10 to ease visual inspection since the spore density in the original suspension was too low for a meaningful comparison. The concentration was done by another centrifugation step (4000 g, 4°C, 10 min), discarding the supernatant and suspending the pellet with 1/10 Milli-Q water of the original volume. The pellet resulting from centrifugation was comprised of three phases, with only the lowest phase at the bottom containing free spores, followed by forespores and cell debris (according to Stier & Kulozik, 2020;Stier et al., 2021). Therefore, to study sporulation success, it was necessary to keep all three phases intact when discarding the supernatant not to distort the result, respectively the ratio of spores to nonsporulated cells by discarding one of the upper phases and not to lose spores, which is to some extent unavoidable with this type of purification. dedicated to cell growth. This may be partly due to the significantly higher nutrient contents in the cultivation media compared to the sporulation media but also due to lower levels of specific ions. The pH value of the media (Table 1) is not expected to be a reason for growth stagnation since the changes were moderate within the incubation time.

| RESULTS AND DISCUSSION
The next step was to examine whether the sporulation media were also suitable for the one-step production process or could lead to similar results as the two-step production process. Since cell growth in our past study (Stier & Kulozik, 2020) also took place in a shake flask and the vegetative cells were only transferred to a bioreactor for sporulation, this experiment was performed exclusively in a shake flask. This provided better comparability between the experiments because vegetative cell growth to sporulation in the bioreactor would have had a significantly different effect on the cultures.
As described above, the cell density in BSM decreased at a certain time of incubation without spores forming. Due to this, we suspected that this medium could not be suitable for producing B. atrophaeus ATCC 9372 spores. We, therefore, extended the comparison and included MSM as another sporulation medium. In the previous cultivation media experiments, this medium was initially not included because it is a medium for obtaining spores using the solid-state method on agar plates (Pruß et al., 2012). To allow submerged cultivation with this medium, the recipe was adjusted by omitting agar-agar. The media were inoculated with 500 µl of B. atrophaeus spores from the same production batch and incubated for 96 h. Within this period, cultivation and sporulation occurred in the same medium without additional intervention. Figure 2 shows representative results of the double determination of spore formation in the respective sporulation media after 96 h of incubation.
As suspected, the spore yield in BSM (Figure 2b) was comparatively low. Only a few free spores could be found in this medium. In addition, a few dead cells (gray rods) were sighted, suggesting that the majority of the cells lysed, as suspected already in the first experiment. Significantly more spores were formed in MSM ( Figure 2c). Although this medium was not intended for submerged spore production in its original recipe, it appears suitable for B.
atrophaeus ATCC 9372. Nevertheless, not all cells sporulated in this medium, and a large proportion died. The medium DSM (Figure 2a) produced the best sporulation efficiency in this comparison. In this medium, the majority of cells appeared to be sporulated, and only a few dead cells were found. The absence of vegetative cells was tested by microscopy of several duplicate samples.
The spore suspensions of these sporulation media were also plated on Plate Count Agar to determine the spore concentration.
Since there were no longer any vegetative cells in the suspensions, the results provided information about the pure number of germinable spores in the respective medium (1 CFU was set equivalent to 1 germinable spore). The spores were not heatactivated before plating. In DSM, an average spore concentration of 1.6 × 10 8 CFU/ml could be observed, in BSM 4.8 × 10 7 CFU/ml, and in MSM 8.4 × 10 7 CFU/ml (these spore concentrations correspond to the original spore suspension before concentration by a factor of 10 for phase-contrast microscopic evaluation). These results confirm the results of the phase-contrast microscopic evaluation that the highest number of spores was formed in DSM. Since the initial cell concentration applied for inoculation was identical in all media, this result shows that DSM produces the best sporulation efficiency among the tested sporulation media.  The results also show that the one-step production process is, in principle, suitable for obtaining spores, depending on the sporulation medium selected. It is notable, though, that an OD 600~3 .9 was achieved in DSM during cultivation, and no decrease in growth or beginning sporulation was observed up to this point. In our previous procedure to produce B. atrophaeus spores (Stier & Kulozik, 2020), we inoculated the bioreactor with an OD 600 of 1.0. Sporulation, therefore, started about 3 h later at a comparatively low cell density.
Nevertheless, we achieved spore yields of 1.1 × 10 9 CFU/ml in this two-step production approach under similar sporulation conditions (30°C, pH 7.2, 30% pO 2 ). Accordingly, overinoculation of vegetative cells from a cultivation medium into the sporulation medium seems to result in a more significant proportion of cells sporulating than in the one-step manufacturing process. In our opinion, this is the only way to explain why, despite a lower cell density in the sporulation medium, we obtained a spore yield increased by a factor of almost 10 in the two-step production process. This difference can be attributed to the best conditions during the cell growth phase in the two-step method achieving high cell numbers at first before entering the sporulation phase, which was also performed under optimal conditions yielding high spore numbers and avoiding losses due to cell death. These results underline our previous recommendations for submerged production of B. atrophaeus spores for use as BI. Although the one-step manufacturing procedure could reduce effort and cost, this is accompanied by significantly lower spore yields.

| CONCLUSIONS
Standardization of BI production is essential to ensure consistent quality and resistance of spores. We have previously recommended a two-step manufacturing process for producing spores for use as BI. However, a one-step manufacturing process would have potential advantages in terms of effort and cost, which would potentially also enable standardization to be uniformly established by and between BI manufacturers. However, since the one-step manufacturing process could obtain only low spore yields, we restate our previous recommendation to prefer the two-step manufacturing process with separated cultivation and sporulation phases. Practical experience in aseptic food and pharmaceutical production shows that standardization of BI manufacturing is essential to reduce uncertainties of sterilization validation tests. An aspect to be addressed in future works is to test the resistance variability between the one-step and the two-step BI production methods. It has already been shown in our previous work (Stier & Kulozik, 2020) that resistance variability can be reduced by a better understanding of the relationship between sporulation conditions and spore resistance. Since suitable media and methods are now available as a result of this work, this will be tested in future experiments.

ACKNOWLEDGMENTS
This IGF Project AiF 19358N of the FEI was supported via AiF within the program for promoting the Industrial Collective Research (IGF) of the German Ministry of Economic Affairs and Energy (BMWi), based on a resolution of the German Parliament. Open Access funding enabled and organized by Projekt DEAL.

CONFLICT OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
All data are provided in full in the results section of this paper, apart from the two-step production data, which are available at https://doi. org/10.3390/molecules25132985.

ETHICS STATEMENT
None required.